Radioresistant alga of the Coccomyxa genus

ABSTRACT

The invention relates to novel algae of the  Coccomyxa  genus, in particular the algae of a novel species called  Coccomyxa actinabiotis , and to the use thereof for metal uptake from aqueous media, and in particular from radioactive media.

RELATED APPLICATIONS

The present application is a U.S. National Phase Application of International Application No. PCT/IB201/050589 (filed Feb. 11, 2011) which claims priority to French Application No. 1000578 (filed Feb, 12, 2010) which are hereby incorporated by reference in their entirety.

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A computer readable text file, entitled “045636-5205_SeqListing_(—)08092012.txt,” created on or about Aug. 7, 2012, with a file size of about 28 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.

The invention relates to novel algae and to the use thereof for metal uptake from aqueous media, and in particular from radioactive media.

Radioactive effluents are produced mainly by nuclear power stations. They are mainly water from spent fuel storage pools, water from decontamination tanks, or else water from cooling circuits of nuclear plants, which ultimately contain radioactive compounds owing to the activation of inactive compounds by radiation, or to the release and dissolution of radioactive compounds. Other sources of radioactive effluents are nuclear medicine, research laboratories using radioactive materials, and also certain non-nuclear industries (for example extraction of rare earths).

Various physical and chemical methods are used to purify the effluents, in particular water, containing radioactive compounds. However, they have high operating and equipment costs, require heavy maintenance and generate large volumes of radioactive waste. Furthermore, their field of application is often limited. For example, ion exchange resins are used to maintain low conductivity in water from nuclear plants. They become loaded with radioactive ions and are stored while awaiting a suitable retreatment procedure when they are saturated, or stored under conditions using toxic or highly reactive compounds.

Moreover, biological methods exist which use, for example, bacteria, fungi, yeasts or plants to purify media contaminated (industrial effluents, natural media, etc.) with toxic products (nonradioactive or radioactive). These methods use living organisms to concentrate and assimilate polluting compounds and to make them less toxic (modification of the chemical form) or non-living biomass and also derivatives originating from living organisms to biosorb pollutants. The biological methods generally have a broader field of application than the physical and chemical methods. They do not require the addition of chemical reagents or products and generally enable less expensive treatment, hence their economic interest.

Plants in particular are good soil or water purifiers since they have an entire system of metabolites, proteins, enzymes, import mechanisms, membrane channels, internal structure, etc., which make them capable, as appropriate, of immobilizing toxic compounds, of chelating them outside or inside the plant, of incorporating them in more or less large amounts via specific or nonspecific import pathways, of suppressing them inside cells, of modifying their speciation so as to make them harmless or less toxic, of making them less soluble, of storing them in nontoxic form in the vacuole, etc.

Studies have shown that certain microorganisms are capable of concentrating metal ions by biosorption, such as Ag, Al, Au, Co, Cd, Cu, Cr, Fe, Hg, Mn, Ni, Pb, Pd, Pt, U, Th, Zn, etc., in dilute solutions (White et al., International Biodeterioration & Biodegradation, 35: 17-40, 1995; U.S. Pat. No. 6,355,172). Biosorption is the capacity of the biomass to bind heavy metals by means of nonselective physicochemical mechanisms via interactions with the functional groups of parietal compounds located at the surface of cells. For example, it has been proposed to use bacteria and mixtures of microorganisms to nonselectively biosorb heavy metals (U.S. Pat. No. 7,479,220; PCT application WO 03/011487).

Other methods use dead biomass or derived compounds originating from the culture of living organisms to depollute media contaminated with metals. The processes involved are biologically inactive, physicochemical mechanisms, such as ion exchange, for example with the polysaccharides present in cell walls, complexation or adsorption.

Biomass derived from algae (for example cell walls) has been used to purify metals contained in liquid effluents (U.S. Pat. No. 4,769,223; PCT application WO 86/07346; U.S. Pat. No. 5,648,313; PCT application WO 2006/081932).

There are few methods which call upon living organisms for the treatment of media polluted with radioactive compounds. Indeed, in the case of water contaminated with radioactive compounds or water located close to radioactive sources, it is necessary to use radiotolerant or radioresistant organisms, which are in addition capable of withstanding the chemical toxicity of the contaminants and of binding the compounds of interest in sufficient amount to be used in the context of an industrial process.

In the natural environment, organisms accumulating radioactive compounds are generally subjected to low radioactivity. For example, immediately after the accident at Chernobyl, for the aquatic media, the external dose rate of ionizing radiation of the reactor cooling tank water did not exceed 100 μGy/h and the maximum cumulative dose over one year was 0.01 Gy in 1986. The dose rate originating from the radionuclides deposited on the sediments in the Pripyat river, located in the 30 km zone around the power plant, increased, for its part, here and there, to 0.4 mGy/h immediately after the accident (Kryshev and Sazykina, Journal of Environmental Radioactivity, 28: 91-103, 1995).

In most cases, the resistance to ionizing radiation of the microorganisms proposed for depolluting materials or effluents contaminated with radioactivity and/or concentrating radioactive compounds has not been tested. This is because they are used to extract U (uranium) and Th (thorium) of which the activity of the main isotopes is low (for example, for ²³⁸U or ²³⁵U, the activity is 0.13 or 0.8 Bq/l for a solution containing 10 μg/l of ²³⁸U or ²³⁵U, respectively.

U.S. Pat. No. 4,320,093 proposes the use of fungi of the Rhizopus genus for extracting uranium or thorium contained in aqueous effluents. Patent GB 1472626 proposes the use of mutant single-cell green algae obtained by X-ray irradiation of single-cell green algae pre-accustomed to uranium, and patent GB 1507003 proposes the use of various microorganisms, in particular the fungus Aspergillus niger, and cyanobacteria of Oscillatoria type, for concentrating the uranium naturally present in sea water. U.S. Pat. No. 7,172,691 proposes the use of live photosynthetic algae of the Chlorella, Scenedesmus, Oocystis and Chlamydomonas genera for concentrating radioactive contaminants, and in particular uranium, from aqueous media containing a concentration of uranium of about 0-20 ppm, which represents an activity of 260 and 1600 Bq/l for ²³⁸U and ²³⁵U, respectively. In comparison, the activity of water from nuclear element storage pools which constitute the living environment of the microalga described hereinafter is approximately 300 000 Bq/l.

The most radioresistant organisms described to date are prokaryotes. The species Deinococcus radiodurans has an extraordinary capacity for resistance to ionizing radiation, grows under irradiations of 60 Gy/h and survives at doses of 15 kGy. However, this bacterium is naturally not very resistant to metals. For example, it does not tolerate cobalt (irrespective of the isotope), which inhibits its growth at 5 ppm, i.e. at approximately 10⁻⁴ mol/l (John et al., Symposium Chemical-biological Interactions In Contaminant Fate, Metal Toxicity In Deinococcus radiodurans, p. 426-428 in Preprints of Extended Abstracts Vol. 40 No. 2, 2000). Its use as a purifier of metals contained in radioactive media therefore requires genetic modifications in order to introduce genes which make it possible to accumulate the metals of interest. Thus, it has been proposed to genetically modify bacteria of the Deinococcus genus so as to express enzymes capable of detoxifying or of metabolizing organic compounds, metals or radionuclides, for the purposes of in situ bioremediation of nuclear waste sites (PCT application WO 01/23526). More recently, bacteria of the Kineococcus radiotolerans species have been isolated and purified from a radioactive waste site of high activity. These bacteria have been described as being capable of degrading organic contaminants in the presence of ionizing radiation, the dose rate of which is greater than 10 Gy/h, and their use for nonselective depollution of radionuclides by biosorption has been proposed (U.S. Pat. No. 7,160,715).

Although they can be used for decontaminating media of which the radioactivity is very high, these two microorganisms have the drawback of being non-autotrophic, and therefore of requiring the external provision of carbonaceous nutrients in order to be able to be used in the form of live cultures. Furthermore, the culture thereof is more sensitive to contamination with other bacteria than that of autotrophic organisms, which, moreover, require a less rich culture medium.

A single form of radiotolerant autotrophic organism has been described (Farhi et al., 20: 104216, 2008). It is a microalga of the class Chlorophyceae, which tolerates ionizing radiation with an LD50 of 6 kGy, whereas, in general, the LD50s for resistance to ionizing radiation of algae are between 30 and 1200 Gy (IAEA, 1976, Effects of ionizing radiation on aquatic organisms and ecosystems. Technical reports series No. 172, International Atomic Energy Agency, Vienna).

The inventors have now isolated another radiotolerant microalga, belonging to the Coccomyxa genus (class Trebouxiophyceae: Pröschold and Leliaert, Unravelling the algae: the past, present, and future of algal systematics, CRC Press, Brodie and Lewis eds., 2007), and have discovered that, not only does this alga exhibit resistance to ionizing radiation that is even greater than that of the microalga described by Farhi et al. (2008, above), but that, in addition, it is capable of taking up and concentrating radioactive or nonradioactive metal ions in solution in an aqueous medium, and that it can grow in a radioactive medium.

This alga represents a new species of Coccomyxa, hereinafter referred to as Coccomyxa actinabiotis.

An axenic culture representative of this new species was deposited according to the treaty of Budapest on Jun. 23, 2009, with the Culture Collection of Algae and Protozoa (CCAP), Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, GB-Oban, Argyll, PA37 1QA, UK, under number CCAP 216/25.

The algae of the Coccomyxa actinabiotis species are characterized in particular in that their genes corresponding to 18S ribosomal RNA-ITS1-5.85 ribosomal RNA-ITS2-26S ribosomal RNA (start) contain a sequence exhibiting at least 95%, and, in order of increasing preference, at least 96%, 97%, 98% or 99% identity with the sequence SEQ ID NO: 1.

The percentage identity indicated above is calculated after alignment of the sequences using the Clustal software (Larkin et al., Bioinformatics, 23, 2947-2948, 2007), on a comparison window consisting of the whole of the sequence SEQ ID NO: 1.

The algae of the Coccomyxa actinabiotis species can also be characterized in that the region corresponding to ITS1-5.8S rRNA-ITS2 exhibits at least 90% identity with the corresponding region of the sequence SEQ ID NO: 1. This threshold was estimated at 90% on the basis of the observations by the inventors, which show a maximum of 81% identity between this region in Coccomyxa actinabiotis and in other Coccomyxae and also a maximum of 88% identity between the ITS1-5.85 rRNA-ITS2 of the other Coccomyxae compared with one another (cf. table III hereinafter).

The sequence of the ribosomal RNA genes of Coccomyxa actinabiotis differs in particular from that of other species of the Coccomyxa genus through the presence, in the region corresponding to the 18S rRNA, of two inserts of approximately 500 bp (inserts represented in italics in FIG. 2). It also differs through the nature of its 18S rRNA gene (FIG. 3), and through that of its ITS1 and ITS2.

Coccomyxa actinabiotis, by virtue of its resistance to ionizing radiation, can grow in a radioactive medium, which allows it to take up and metabolize radioactive compounds other than metals (for example ³H or ¹⁴C), in addition to being able to take up and concentrate radioactive or nonradioactive metal ions in solution in an aqueous medium.

Consequently, the subject of the present invention is the use of green algae of the Coccomyxa genus, and in particular of the Coccomyxa actinabiotis species defined above, for taking up at least one element chosen from the metals Ag, Co, Cr, Zn, Mn, Sb, Cs, Ni, Fe, actinides, or lanthanides, whether they are radioactive or not, or the radioisotopes ¹⁴C and ³H, from an aqueous medium containing said metal or said radioisotope in solution.

More particularly, the subject of the present invention is a method for taking up at least one element chosen from the radioactive or nonradioactive metals Ag, Co, Cr, Zn, Mn, Sb, Cs, Ni, Fe, actinides or lathanides, or the radioisotopes ¹⁴C and ³H from an aqueous medium containing said element in solution, characterized in that said uptake is carried out by incubating green algae of the Coccomyxa genus in said aqueous medium.

Advantageously, said element is a metal chosen from Ag, Co, and the actinide U.

Also advantageously, said element is ¹⁴C. The amount of ¹⁴C in the waste from nuclear plants in France has only been regulated individually for the last few years. However, ¹⁴C is the principal radioactive pollutant emitted into the environment by nuclear plants, after tritium. For example, the basic nuclear plants of the Tricastin nuclear power station (4 sections of 900 MW) discharged 170 GBq of ¹⁴C into the atmosphere and 15.9 GBq of ¹⁴C in liquid effluents in 2009 (EDF Annual Report 2009). Reprocessing factories which use the Purex® process (for example, AREVA The Hague) discharge into the environment approximately 600 GBq/year of ¹⁴C for reprocessing all of the fuel of a 1 GWe reactor (Toxicologie nucléaire environnementale et humaine [Environmental and human nuclear toxicology], eds Tec and Doc, Lavoisier, 2009).

According to one preferred embodiment of the present invention, said aqueous medium is a radioactive medium, i.e. a medium in which said algae are subjected to a dose rate that can range from a few μGy/h to 1 kGy/h. According to one preferred arrangement of this embodiment, the element to be taken up is a metal chosen from those indicated above, in the form of a radioactive isotope, or in the form of a mixture of isotopes.

The incubation time of the algae in the aqueous medium can vary in particular according to, firstly, the element(s) concerned and, secondly, the nature of the aqueous medium from which the uptake must be carried out. It will generally be at least 1 hour, and may range up to several months, or even several years. For example, if it is desired to take up only Ag, an incubation time of approximately 1 hour may be sufficient to take up the major part thereof, and of approximately 10 to 20 hours to take up virtually all thereof; if other metals are to be taken up, a longer incubation time, of at least 3 to 5 days, and advantageously of at least one week, may be used. As another example, if it is desired to take up ¹⁴C, an incubation time of 3 h, under the optimized conditions proposed in example 4 hereinafter, will be sufficient to take up the major part thereof.

The maximum incubation time that can be envisaged will in fact depend mainly on the capacity for growth and survival of the algae in the aqueous medium.

In the presence of light and carbon dioxide (introduced by contact with the ambient air, agitation of the cultures or bubbling), the algae of the Coccomyxa genus, and in particular of the Coccomyxa actinabiotis species, can grow and live for very long periods of time in weakly mineralized water (conductivity 1 to 1.5 μS/cm) at a pH of 5 to 6 and a temperature of 20 to 30° C., and in demineralized water (conductivity of 0.05 μS/cm), they can grow and live for 3 to 4 weeks. Since these green algae need light to carry out photosynthesis and produce their organic matter, their growth stops when they are placed in the dark.

Thus, according to one embodiment of the method in accordance with the present invention, the growth of the green algae of the Coccomyxa actinabiotis species can be controlled by controlling the illumination of the aqueous medium comprising said algae.

The Coccomyxa actinabiotis algae can also grow and live for several years in a weakly radioactive medium, where they are subjected to an irradiation of less than or equal to 0.15 mGy/h. They can withstand an irradiation of 1 Gy/h for up to approximately 1 month. They can also withstand an irradiation of 300 Gy/h for approximately a day and an irradiation of 100 Gy/h for approximately 3 days. In a very highly radioactive medium, they can withstand an irradiation of 1 kGy/h for up to 20 hours; if they are then transferred into a nonradioactive medium, they recover their growth capacity in less than two weeks. By way of indication, the limit of the range of low dose rates below which no biological effect has been detected can be placed at around 1 mSv/h (i.e. 1 mGy/h for γ-radiation). A low dose corresponds to a dose of less than 10 mSv/h. A dose rate greater than 1 mSv/h produces dangerous biological effects. For doses greater than 10-100 mSv, statistically observable effects appear. A high dose corresponds to an irradiation greater than 1 Gy, the value starting from which a deterministic effect begins to become apparent.

For implementing the process in accordance with the invention, the algae can be used in suspension in the aqueous medium from which the uptake is to be carried out, with shaking so as to avoid agglomeration thereof. They can also be attached to a solid support which is smooth, porous, or in the form of beads, placed in said aqueous medium.

The process can be transported, i.e. the algae are brought into contact with the medium to be depolluted in a chamber separate from the nuclear plant, or implemented in situ; in the latter case, the algae are then implanted directly in the medium to be depolluted.

In the case of an in situ uptake or decontamination process, the algae can reside in the plant as long as they do not impair operations. By way of indication, their growth can be controlled by the intensity of illumination (darkness or weak illumination), or the choice of the wavelength of the lamps (for example, yellow-green non-actinic light). Filtration of the water also makes it possible to control their growth by taking up the algae in suspension in the water. If it proves to be necessary to remove them, definite destruction of the algae may be accompanied by release of the metals, and will therefore have to be preferentially carried out by section in the plants, with recovery of the effluents containing a concentration of metals.

In the case of a transported process, at the end of the incubation, the algae having taken up the metals are harvested by conventional means (filtration, decanting, centrifugation, etc.). They can then be eliminated as waste, optionally after having been dried and/or burnt, without prior extraction of the metals that they contain.

Advantageously, the metals taken up by the algae can be recovered in order to recycle these metals.

This recovery can be carried out by any appropriate means. It is, for example, possible to non-destructively recover at least a part of the metals taken up by the algae, thereby making it possible, where appropriate, to reuse said algae, in particular for metals such as Ag and Cr, which, after uptake, remain largely at the level of the mucilage and of the cell surface. This recovery can be carried out by placing the algae in the presence of a complexing or acid solution. Thus, it can in particular be carried out for metals such as Ag, by treatment of the algae in an acidic medium, at a pH of from 1 to 2.5, preferably from 1.5 to 2, for 1 to 2 days.

For other metals, such as Cr, but also for the portion of metals such as Co which is bound at the level of the mucilage and of the cell surface, it can also be carried out by means of a treatment with a chelating agent, such as EDTA, at 0.1 mol/l for 1 h up to 3 days, preferably for approximately 1 day.

For metals such as Co which, after uptake by the algae, are sequestered inside the cells, the recovery can be carried out by treating the algae in an acidic medium, at a pH of 0.5, for 2 to 10 days, but without any guarantee of the subsequent viability of the cells.

The metals can also be recovered after destruction of the algae. This destruction can, for example, be carried out by lysis of the algae. It can also be carried out by incineration of the algae.

The process in accordance with the invention can be implemented in all cases where it is desired to extract radioactive or nonradioactive metals, and in particular those mentioned above, from an aqueous medium, for the purposes of mining operations, or of depollution of aqueous effluents containing metals, in particular of radioactive effluents.

In addition to their strong capacity for specifically taking up and concentrating the metals mentioned above (Ag, Co, Cr, Zn, Mn, Sb, Cs, Ni, Fe, actinides or lanthanides, whether they are radioactive or nonradioactive), the algae of the Coccomyxa genus, and in particular of the Coccomyxa actinabiotis species, also have properties of nonspecific uptake of other metals, in particular owing to their extracellular mucilage consisting of polysaccharides, which have the property of complexing cations.

The process in accordance with the invention is therefore particularly suitable for depolluting and for decontaminating aqueous media or soils (peat bogs, marshes) contaminated with radioactive or nonradioactive metals, and more particularly radioactive media, such as the water from storage pools or else the light water from secondary cooling circuits of nuclear power stations or reactors, or the effluents from nuclear power stations discharged into the environment.

The present invention can be implemented not only with single-cell green algae of the Coccomyxa actinabiotis species, but also with a mixture of microorganisms comprising single-cell green algae of the Coccomyxa actinabiotis species and at least one microorganism, in particular a bacterium, a fungus, a yeast, another single-cell alga and/or a multicellular plant, preferably radioresistant or radiotolerant, capable of concentrating metal ions in solution and/or of taking up and metabolizing radioactive compounds other than metals (for example, ³H or ¹⁴C). The multicellular plants and microorganisms that can be used in combination with the single-cell green algae of the Coccomyxa actinabiotis species are especially those mentioned above, in particular those which are radioresistant or radiotolerant. In the case where the present invention is applied to a radioactive aqueous medium, the incubation time of the mixture of microorganisms will depend on the individual resistance of the microorganisms making up the mixture. Similarly, the culture conditions may be adjusted in order to promote the growth of one or more microorganisms making up the mixture.

The present invention will be understood more clearly from the further description which follows, which refers to examples illustrating the isolation and the characterization of the Coccomyxa actinabiotis species, and also its use for decontaminating a radioactive aqueous medium. The examples are illustrated by means of the following figures:

FIGS. 1A and 1B represent photographs of microalgae according to the invention, observed by photon microscopy (Zeiss binocular Axioplan 2 microscope, magnification 1000 and 3000 for FIGS. 1A and 1B, respectively). FIG. 1B shows the joining of two flagellated cells.

FIG. 2 shows the sequence of the 18S rRNA-ITS1-5.85 rRNA-ITS2-26S rRNA ribosomal DNA genes (first 500 bases) of the Coccomyxa actinabiotis microalgae.

FIG. 3 shows the comparison of the sequences corresponding to the small subunit ribosomal RNA (18S rRNA) of the microalga CCAP 216/25, and of the other Coccomyxa species listed in the databases (after having taken away the 2 inserts specific for the sequences SEQ ID NO: 1), carried out by multisequence alignment by means of the Clustal W2 2.0.12 software.

FIG. 4 shows the percentage mortality of Coccomyxa actinabiotis as a function of the dose of irradiation in gray (Gy).

FIG. 5 illustrates the binding and concentrating of metals by Coccomyxa actinabiotis. The concentrating factors, defined as the ratio of the concentration of the metal adsorbed or absorbed in the microalgae (in atoms/g of fresh matter) to the concentration of the metal in the water (atoms/ml), obtained for the metals ^(110m)Ag, ⁶⁰Co, ⁵¹Cr, ⁶⁵Zn, ⁵⁴Mn and ¹²⁴Sb, are represented.

FIGS. 6 to 8 show the kinetics for binding and for incorporation of metals, silver (FIGS. 6 and 7) and cobalt (FIG. 8), by the Coccomyxa actinabiotis microalgae, with (FIG. 7) or without (FIGS. 6 and 8) mucilage. The amounts of metal adsorbed-absorbed, as percentage, are represented as a function of exposure time (in hours). FIG. 6A: solution containing 110 μg/l of silver ions; FIG. 6B: solution containing 5.5 mg/l of silver ions; FIG. 7A: solution containing 1.1 mg/l of silver ions; FIG. 7B: solution containing 55 mg/l of silver ions; FIG. 8A: 19 μg/l of cobalt ions; FIG. 8B: 0.94 mg/l of cobalt ions; FIG. 8C: 4.8 mg/l of cobalt ions.

FIG. 9 shows the incorporation of ¹⁴C by a culture of Coccomyxae actinabiotis. The percentage of ¹⁴C in hydrogen carbonate form, in the pellet (triangles), which contains the algae, and in the culture supernatant (diamonds), is represented as a function of time, in hours.

FIG. 10 represents the concentration of silver-110m, expressed in kBq/l, in a radioactive element storage pool, as a function of time (in days), in the presence of Coccomyxae actinabiotis.

EXAMPLE 1 Isolation and Characterization of Coccomyxa actinabiotis

The microalga was harvested from a spent fuel storage pool of a nuclear reactor. The water contained in this pool has a pH of 5.2 to 5.8 and a conductivity of from 1 to 1.5 μS/cm, is in contact with the ambient air and contains dissolved radioactive elements. Its temperature ranges between 23 and 30° C. and is on average 25° C. The radiological activity in the pool is very variable depending on the measuring points, low to very high. The gamma activity can reach several thousand Gy/h upon contact with spent fuel elements.

The presence of films of green organic matter was observed on the walls and various surfaces of this pool. Samples were taken and when observed under a microscope they showed that this was a single-cell green microalga.

Culture Conditions

The samples taken were stored and placed in culture in light, at a pH of from 5 to 6.5 and a temperature of 23° C., in sterile Erlenmeyer flasks closed with porous stoppers allowing gas exchange.

The following media were tested:

-   -   BBM culture medium (Bold's Basal Medium, Sigma), pure or diluted         in demineralized water. BBM medium is conventionally used for         culturing green algae;     -   BG11 culture medium (Rippka et al., “The Prokaryotes”, vol. 1:         212-220, 1979; Sigma). This medium is conventionally used for         culturing cyanobacteria;     -   weakly mineralized water (conductivity from 1 to 1.5 μS/cm), or         demineralized water (conductivity of 0.05 μS/cm).

The composition of the BBM and BG11 culture media is indicated in table I hereinafter:

TABLE I BBM BG 11 Constituents in g/L NaNO₃ 0.25 1.5 KH₂PO₄ 0.175 K₂HPO₄ 0.075 K₂HPO₄•3H₂O 0.04 MgSO₄•7H₂O 0.075 0.075 FeSO₄•7H₂O 0.005 CaCl₂•2H₂O 0.025 0.036 NaCl 0.025 Na₂EDTA 0.01 KOH 0.006 Citric acid 0.006 Ferric ammonium citrate 0.006 Trace elements in mg/L H₃BO₄ 12.86 2.86 MnCl₂•4H₂O 1.81 1.81 ZnSO₄•7H₂O 0.222 0.222 Na₂MoO₄•2H₂O 0.39 0.39 CuSO₄•5H₂O 0.079 0.079 Co(NO₃)₂•6H₂O 0.049 0.049 pH 6.4 6.5

In the BG11 and BBM media, the microalgae grow with a similar exponential growth phase in the two media.

In the weakly mineralized water, or in the demineralized water with a conductivity of 0.05 μS/cm, the microalgae grow with a rapid growth phase in the short term, similar to that obtained with the BG11 and BBM media, but in the demineralized water with a conductivity of 0.05 μS/cm, their health deteriorates after 3 to 4 weeks, probably due to the depletion of reserves.

Algae were placed in culture on a solid agar BBM culture medium. Circular colonies were thus isolated, and then plated out individually on agar culture medium. This operation was repeated six times in order to obtain a pure culture originating from a single cell.

A sample of this culture (hereinafter referred to as microalga CCAP 216/25) was deposited according to the treaty of Budapest, on Jun. 25, 2009, with the Culture Collection of Algae and Protozoa (CCAP), Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, GB-Oban, Argyll, PA37 1QA, UK, under number CCAP 216/25.

Morphological and Biochemical Characteristics

The microalgae isolated are single-cell microalgae. The cells, observed by photon microscopy and by confocal microscopy, are nucleated and oval in shape. Their average length is 6.8±0.9 μm and their average width is 3.8±0.6 μm. However, depending on their growth stage, it is possible to observe longer average lengths, up to 9-10 μm, or smaller ones, of about 5 μm, in particular just after division.

They contain a chloroplast (perhaps several) which contains chlorophyll and which is the site of photosynthesis. A protein granule, the pyrenoid, occupies part of the chloroplast. It is involved, inter alia, in the synthesis of starch. Other organelles, in particular the vacuole, occupy the rest of the cell.

In its vegetative form, the cell does not have mobility. Sometimes, certain individuals have two locomotor flagellae. They are either sexual cells or gametes, which perform reproduction, or zoospores which are involved in multiplication so as to allow dissemination of the species. The formation of these two types of cells is provided by the division of an initial cell or mother cell.

In culture on agar medium, the cells remain grouped in discoid aggregates.

Some differences distinguish the cells freshly sampled from the storage pool from those cultured for one month in BBM medium. The first have large storage granules, are mostly agglomerated to one another, held by a mucilage consisting of polysaccharides, and the mobility of their flagellated cells is reduced. When the algae cultured in the BBM medium are returned to radioactive and nutritive stress conditions, they go back to the morphology of those isolated from the storage pool water.

The second are more pigmented (bright green), generally well individualized, and their flagellated cells are very active when the culture is regularly stirred.

The cytological and behavioral modifications of this microalga in the hostile medium from which it was isolated can therefore be perceived as an adaptation to the particular physicochemical conditions (presence of ionizing radiation, presence of radioactive metals inducing oxidative and ionizing stress; lack of mineral salts, responsible for nutritive stress).

FIGS. 1A and 1B represent photographs of these microalgae observed by photon microscopy, one month after the introduction of a sample originating from the pool of the reactor in a BBM culture medium.

Starch, located in the chloroplast, can be revealed by staining with lugol (I₂+IK=iodinated water). A positive response to this reaction (brown to purplish blue coloration) makes it possible to categorize this microalga among the green algae (=chlorophyta).

The UV-visible absorption spectrum for this organism shows the presence of chlorophyll a (absorption peak at 663 nm), chlorophyll b (absorption peak at 647 nm) and carotene (absorption peak at 470 nm).

It is therefore a single-cell, eukaryote and freshwater taxon of green algae. It belongs to the kingdom Protista with affinity to plants, and therefore to the Protophyta. It lies in the division Chlorophyta or Green Algae since it contains chlorophylls a and b and carotene as photosynthetic pigments and its reserves are starch.

Amplification and Sequencing of the Ribosomal DNA Genes

The total DNA of microalga A isolated as described above was extracted by the method of Newman et al. (Genetics, 126: 875-888, 1990).

The region of the genome covering the 18S rRNA-ITS1-5.8S rRNA-ITS2-26S rRNA (first 500 bases) ribosomal DNA genes was amplified by PCR and sequenced.

The primers used are EAF3: TCGACAATCTGGTTG ATCCTGCCAG (SEQ ID NO: 2) and ITS055R: CTCCTTGGTC CGTGTTTCAAGACGGG (SEQ ID NO: 3), conventionally used for amplifying microalga rRNA genes.

The amplification products obtained using the DNA isolated from two independent cultures are of 4 kb (i.e. 1 kb more than what is conventionally described for microalgae). The sequence of these amplification products is represented in FIG. 2, and also in the appended sequence listing under number SEQ ID NO: 1. This sequence contains 2 inserts of approximately 500 base pairs which are indicated in bold italics in FIG. 2 and which are specific to the microalga CCAP 216/25.

The BLASTN algorithm (Altschul et al. Nucleic Acids Research, 25: 3389-3402, 1997) was used to search, in the databases, for the sequences of ribosomal RNA genes exhibiting maximum identity with the sequence SEQ ID NO: 1. In order to carry out this search, the two sequences of approximately 500 base pairs which are indicated in bold italics in FIG. 2 were withdrawn. This search revealed that the species characterized closest to the microalga CCAP 216/25 belong to the Coccomyxa genus. The sequence SEQ ID NO: 1 contains, however, 2 specific inserts of approximately 500 base pairs, which are not found in the algae of the Coccomyxa genus that have been characterized up until now.

The comparison of the sequences corresponding to the ribosomal small subunit RNA (18S rRNA) of the CCAP 216/25 microalga, and of the other Coccomyxa species listed in the databases (performed after having withdrawn the two specific inserts of the sequence SEQ ID NO: 1) was carried out by multiple sequence alignment by means of the ClustalW2 2.0.12 software. The sequence alignment is presented in FIG. 3. Table II below gives the results of this sequence comparison. The sequences corresponding to the 18S rRNAs, which are compared in pairs, are indicated in the SEQ ID A and SEQ ID B columns, by reference to their number in the appended sequence listing.

TABLE II SEQ ID A Name Length (nt) SEQ ID B Name Length (nt) % identity 4 CCAP 216/25 2354 5 Coccomyxa peltigerae 1783 97 4 CCAP 216/25 2354 6 Coccomyxa chodatii 1783 98 4 CCAP 216/25 2354 7 Coccomyxa sp flensburg 1778 97 4 CCAP 216/25 2354 8 Coccomyxa glaronensis 1797 96 4 CCAP 216/25 2354 9 Coccomyxa sp CPCC 1797 96 4 CCAP 216/25 2354 10 Chlamydomonas_sp 1558 92 5 Coccomyxa peltigerae 1783 6 Coccomyxa chodatii 1783 99 5 Coccomyxa peltigerae 1783 7 Coccomyxa sp flensburg 1778 96 5 Coccomyxa peltigerae 1783 8 Coccomyxa glaronensis 1797 96 5 Coccomyxa peltigerae 1783 9 Coccomyxa sp CPCC 1797 96 5 Coccomyxa_peltigerae 1783 10 Chlamydomonas_sp 1558 92 6 Coccomyxa chodatii 1783 7 Coccomyxa sp flensburg 1778 96 6 Coccomyxa chodatii 1783 8 Coccomyxa glaronensis 1797 96 6 Coccomyxa chodatii 1783 9 Coccomyxa sp CPCC 1797 97 6 Coccomyxa_chodatii 1783 10 Chlamydomonas_sp 1558 92 7 Coccomyxa sp flensburg 1778 8 Coccomyxa glaronensis 1797 99 7 Coccomyxa sp flensburg 1778 9 Coccomyxa sp CPCC 1797 99 7 Coccomyxa_sp_flensburg 1778 10 Chlamydomonas_sp 1558 91 8 Coccomyxa glaronensis 1797 9 Coccomyxa sp CPCC 1797 99 8 Coccomyxa_glaronensis 1797 10 Chlamydomonas_sp 1558 91 9 Coccomyxa sp CPCC 1797 10 Chlamydomonas sp 1558 91

This sequence comparison shows that the species closest to the CCAP 216/25 strain are Coccomyxa chodatii strain SAG 216-2 (SEQ ID NO: 6), Coccomyxa peltigerae strain SAG 216-5 (SEQ ID NO: 5), Coccomyxa sp. Flensburg fjord 2 (EU127471), Coccomyxa glaronensis strain CCALA 306 (AM167525) and Coccomyxa sp. strain CPCC 508 (AM981206) with, respectively, 98%, 97%, 97%, 96% and 96% sequence identity. These strong identity scores obtained for the CCAP 216/25 strain compared with the Coccomyxa genus are close to those obtained after comparison of the sequences of the Coccomyxae with one another and far from the score obtained for the sequence comparison with a single-cell microalga belonging to another genus (Chlamydomonas sp. CCMP681) (EF106784) (cf. FIG. 3). This indicates that the CCAP 216/25 strain belongs to the Coccomyxa genus.

Moreover, the comparison of the sequences of the ITS region of the CCAP 216/25 strain with those of the other Coccomyxae was also carried out. Table III below gives the results of this sequence comparison. The sequences of the ITS1-5.85 rRNA-ITS2 regions which are compared in pairs are indicated in columns I and II. The sequences of the Coccomyxa actinabiotis (CCAP 216/25), Coccomyxa chodatii SAG 216-2 and Coccomyxa peltigerae SAG 216-5 strains are indicated by reference to their number in the appended sequence listing, namely, respectively, SEQ ID NOS 11, 12 and 13. For the following strains, of which the sequences of the ITS1-5.8S rRNA-ITS2 regions are accessible in the GenBank database, the corresponding accession numbers are also indicated in table III:

-   -   AY293964: Coccomyxa peltigerae var. variolosae     -   AY293965: Coccomyxa solarinae var. croceae     -   AY293966: Coccomyxa solarinae var. bisporae     -   AY293967: Coccomyxa solarinae var. saccatae     -   AY293968: Coccomyxa chodatii     -   AY328522: Coccomyxa peltigerae strain SAG 216-5     -   AY328523: Coccomyxa subellipsoidea strain SAG 216-13     -   AY328524: Coccomyxa rayssiae strain SAG 216-8     -   U66945: Chlamydomonas callosa     -   U66956: Dunaliella tertiolecta     -   AF376740: Pandorina morum

TABLE III I Length (nt) II Length (nt) % identity SEQ ID 11 761 SEQ ID 12 617 81 SEQ ID 11 761 SEQ ID 13 708 79 SEQ ID 11 761 AY293964 610 78 SEQ ID 11 761 AY293965 650 73 SEQ ID 11 761 AY293966 651 73 SEQ ID 11 761 AY293967 651 73 SEQ ID 11 761 AY293968 619 76 SEQ ID 11 761 AY328522 725 73 SEQ ID 11 761 AY328523 702 75 SEQ ID 11 761 AY328524 696 76 SEQ ID 11 761 U66945 625 43 SEQ ID 11 761 U66956 598 45 SEQ ID 11 761 AF376740 626 27 SEQ ID 12 617 SEQ ID 12 708 87 SEQ ID 12 617 AY293964 610 83 SEQ ID 12 617 AY293965 650 82 SEQ ID 12 617 AY293966 651 83 SEQ ID 12 617 AY293967 651 83 SEQ ID 12 617 AY293968 619 92 SEQ ID 12 617 AY328522 725 84 SEQ ID 12 617 AY328523 702 80 SEQ ID 12 617 AY328524 696 94 SEQ ID 12 617 U66945 625 38 SEQ ID 12 617 U66956 598 31 SEQ ID 12 617 AF376740 626 26 SEQ ID 13 708 AY293964 610 97 SEQ ID 13 708 AY293965 650 99 SEQ ID 13 708 AY293966 651 99 SEQ ID 13 708 AY293967 651 99 SEQ ID 13 708 AY293968 619 88 SEQ ID 13 708 AY328522 725 94 SEQ ID 13 708 AY328523 702 80 SEQ ID 13 708 AY328524 696 86 SEQ ID 13 708 U66945 625 37 SEQ ID 13 708 U66956 598 51 SEQ ID 13 708 AF376740 626 34 AY293964 610 AY293965 650 97 AY293964 610 AY293966 651 97 AY293964 610 AY293967 651 97 AY293964 610 AY293968 619 83 AY293964 610 AY328522 725 96 AY293964 610 AY328523 702 78 AY293964 610 AY328524 696 85 AY293964 610 U66945 625 41 AY293964 610 U66956 598 50 AY293964 610 AF376740 626 48 AY293965 650 AY293966 651 99 AY293965 650 AY293967 651 99 AY293965 650 AY293968 619 88 AY293965 650 AY328522 725 98 AY293965 650 AY328523 702 80 AY293965 650 AY328524 696 85 AY293965 650 U66945 625 37 AY293965 650 U66956 598 45 AY293965 650 AF376740 626 34 AY293966 651 AY293967 651 99 AY293966 651 AY293968 619 88 AY293966 651 AY328522 725 98 AY293966 651 AY328523 702 79 AY293966 651 AY328524 696 85 AY293966 651 U66945 625 37 AY293966 651 U66956 598 51 AY293966 651 AF376740 626 34 AY293967 651 AY293968 619 88 AY293967 651 AY328522 725 98 AY293967 651 AY328523 702 79 AY293967 651 AY328524 696 85 AY293967 651 U66945 625 37 AY293967 651 U66956 598 51 AY293967 651 AF376740 626 34 AY293968 619 AY328522 725 87 AY293968 619 AY328523 702 82 AY293968 619 AY328524 696 96 AY293968 619 U66945 625 37 AY293968 619 U66956 598 40 AY293968 619 AF376740 626 37 AY328522 725 AY328523 702 83 AY328522 725 AY328524 696 88 AY328522 725 U66945 625 37 AY328522 725 U66956 598 51 AY328522 725 AF376740 626 34 AY328523 702 AY328524 696 84 AY328523 702 U66945 625 38 AY328523 702 U66956 598 36 AY328523 702 AF376740 626 35 AY328524 696 U66945 625 37 AY328524 696 U66956 598 31 AY328524 696 AF376740 626 26 U66945 625 U66956 598 58 U66945 625 AF376740 626 59 U66956 598 AF376740 626 51

This sequence comparison shows a 73% to 81% identity of the sequence of the CCAP 216/25 strain compared with the other Coccomyxae strains referenced, of the same order as the scores obtained for the comparison of the ITS region of the referenced Coccomyxae with one another (78% to 99%), and very far from that obtained for the comparison with other genera (about from 30 to 50%).

Furthermore, it is found that the sequence corresponding to the 18S rRNA of the CCAP 216/25 strain is different than that of all the Coccomyxae referenced up until now, in particular in that it has two insertions of approximately 500 base pairs.

The latter two points indicate that it is a Coccomyxa species different than all those referenced to date.

These results therefore indicate that the microalga isolated belongs to the Coccomyxa genus, but that its DNA differs sufficiently from that of the other species of Coccomyxae listed in the databanks, in particular by virtue of its two insertions in the 18S rRNA DNA and ITS1 and ITS2 for it to be considered that this is a new species, which will be named herein Coccomyxa actinabiotis.

Resistance to Abiotic Stresses

Coccomyxa actinabiotis is resistant to desiccation, to cold and to the dark for 48 h.

It is also resistant to pure heavy water for 48 h (respiration and photosynthesis of algae exposed for 24 h and 48 h to 50%, 80% and 100% D₂O, in the dark, and their recovery after 24 h in a normal medium and also respiration and photosynthesis of algae exposed to 24 h to light in 50% D₂O shows that the heavy water has, under these conditions, no effect on these physiological parameters).

This resistance to heavy water makes it possible to propose the use of Coccomyxa actinabiotis (and more generally of algae of the Coccomyxa genus) for the synthesis of deuterated compounds.

Coccomyxa actinabiotis is also resistant to acetone, to alcohol, to acids and to ultrasound.

The resistance to ionizing radiation of the algae was tested by exposing them to various doses of gamma radiation derived from spent fuel elements of which the irradiation is decreasing. The mortality after irradiation was determined by vital staining (neutral red).

Coccomyxa actinabiotis is resistant in the long term (more than about ten years) to moderate irradiation (150 μGy/h).

It withstands exposure for several days to a dose rate of 300 Gy/h and for a few hours to a dose rate of 3000 Gy/h.

It also withstands exposure for 20 h to a flow rate of 1 kGy/h.

FIG. 4 shows the percentage mortality of Coccomyxa actinabiotis as a function of the dose of irradiation. The dose of ionizing radiation which causes the death of half the population is more than 10 kGy (45% mortality at 10 kGy).

Coccomyxa actinabiotis also remarkably withstands exposure to 20 kGy (80%±5% mortality, i.e. 20%±5% survivors). In the case of this strong irradiation at 20 kGy, it recolonizes the medium in a few weeks. In four weeks, the culture has recovered a third of its initial photosynthetic activity before irradiation, and it recovers in two weeks a population density equal to that of nonirradiated or weakly irradiated samples.

EXAMPLE 2 Concentrating of Radioactive Metals by Coccomyxa actinabiotis

Binding and Concentrating of Metals

The microalgae freshly sampled from the storage pool (approximately 10 milligrams of fresh mass) were placed in the presence, under illumination (200 lux), for 1 year, of regularly renewed demineralized water, at pH 5.5, with a conductivity of 1.2 μS/cm, containing for example 400 Bq/l of ⁶⁰Co, 1100 Bq/l of ^(110m)Ag, 1700 Bq/l of ¹²⁴Sb, 5000 Bq/l of ⁵¹Cr, 400 Bq/l of ⁶⁵Zn, 300 Bq/l of ⁵⁴Mn. The water is radioactive essentially owing to the presence of tritium (approximately 300 000 Bq/l) and of ¹⁴C (approximately 20 000 Bq/l). This composition is similar to that of the water from spent fuel storage pools. The algae multiplied slowly during this contact time.

The concentrations of the radionuclides were measured at equilibrium in the waters and in the algae by γ-spectrometry.

The concentration factors for each metal (^(110m)Ag, ⁶⁰Co, ⁵¹Cr, ⁶⁵Zn, ⁵⁴Mn and ¹²⁴Sb), defined as the ratio of the concentration of the metal adsorbed or absorbed in the microalgae (in atoms/g of fresh matter) to the concentration of the metal in the water (atoms/ml), were thus obtained.

The results are illustrated by FIG. 5.

These results show that the microalgae very strongly concentrate silver and cobalt (concentration factor=450 000 and 40 000, respectively), and also chromium to a lesser extent (concentration factor=6000). The least concentrated elements also have a significant concentration factor (concentration factor=6000, 3000, 1200 and 200 for chromium, zinc, manganese and antimony, respectively).

Another experiment was carried out by placing 250 mg of fresh mass of microalgae originating from cultures in BBM medium in the presence of 100 mm of demineralized water which has a composition similar to that of the water from the spent fuel storage pools, namely pH 5.5, conductivity 1.2 μS/cm, containing 280 Bq/l of ⁶⁰Co, 530 Bq/l of ⁵⁸Co, 66 Bq/l of ^(110m)Ag, 1460 Bq/l of ¹²⁴Sb, 1180 Bq/l of ⁵¹Cr, 120 Bq/l of ⁶⁵Zn, Bq/l of ⁵⁴Mn and also tritium (260 000 Bq/l) and ¹⁴C (10 000 Bq/l). After one day of culture, the percentage of each isotope fixed is 100%, 100%, 94%, 94%, 90%, 48% and 30% for ^(110m)Ag, ⁶⁵Zn, ⁶⁰Co, ⁵⁸Co, ⁵⁴Mn, ⁵¹Cr and ¹²⁴Sb, respectively.

Extraction of the Metals from the Microalgae

Experiments in which the metals bound by the algae are solubilized were carried out by incubating these algae (70 mg of fresh matter) in 5 ml of demineralized water, or of demineralized water supplemented with HCl at various concentrations. In neutral medium, the metals are not extracted.

The cations most retained are ⁶⁰Co, ^(108m)Ag and ^(110m)Ag. They remain bound to the cell for low concentrations of HCl (pH 4.5 to 2.5). They begin to be released exponentially below a threshold pH of 2.5, Co being the most solidly bound since it is at most 35% extracted after 6 days of exposure at pH 0.5, whereas Ag is 90% extracted under the same conditions. At pH 1.5, 10% of Ag and 2% of Co are extracted after 6 days of exposure. For ⁵¹Cr, there is no threshold pH, it is extracted from pH=4.5 linearly up to a maximum of 25% at pH=0.5 after 6 days of exposure.

Other experiments were carried out in the presence of a 0.1 M solution of EDTA with a pH of 4.5. Under these conditions, 35% of the ⁵¹Cr contained in the algae, but only 1% of the ⁶⁰Co and less than 0.5% of ^(108m)Ag and ^(116m)Ag, are extracted after 2 days of exposure.

These experiments show that Cr remains predominantly at the surface, whereas Co is probably predominantly sequestered inside the cells and Ag is distributed between the surface and the inside of the cells.

EXAMPLE 3 Kinetics for Binding of Metals, Resistance to their Toxicity and Amounts Bound

Kinetics of Binding and Incorporation of Ag and Co

The kinetics of binding of metals by the Coccomyxa actinabiotis microalgae, and also the amounts adsorbed-absorbed, were determined in the case of a monovalent cation, Ag⁺, and of a divalent cation, Co²⁺.

Silver

Two series of experiments were carried out: one with algae having a coating of mucilage, the thickness of which, evaluated by optical microscopy, is about 1 μm or more (“algae with mucilage”), the other with algae cultured in BBM medium and having little or no mucilage (“algae without mucilage”).

20 mg of algae (compacted fresh mass) and 90 ml of solution of Ag⁺ at various concentrations are introduced into Erlenmeyer flasks. The 20 mg of algae are prepared as follows: sample taken from a round-bottomed culture flask, centrifugation for 20 minutes at 3000 rpm, washing twice in demineralized water (suspension, centrifugation). The culture medium is thus removed and there is no risk of it complexing the silver.

The algae without mucilage are brought into contact with solutions containing 0.11 mg/L and 5.5 mg/L of Ag⁺.

The algae with mucilage are brought into contact with solutions containing 22 μg/L, 1.1 mg/L and 55 mg/L of Ag⁺.

The Erlenmeyer flasks are placed on illuminated shakers and samples of 5 ml are taken for various exposure times in order to monitor the binding kinetics for each of the initial concentrations of silver. The samples are centrifuged for 10 minutes at 4500 rpm in order to separate the algae which have concentrated the metal. The metal content is then analyzed in the pellet (algae) and in the supernatant.

The results are illustrated in FIG. 6 for the algae without mucilage, and in FIG. 7 for the algae with mucilage.

For the algae with or without mucilage, very rapid silver absorption kinetics are observed in the first hours, followed by a plateau.

At the plateau, the algae without mucilage bind all the silver if its initial concentration is 0.11 mg/l and bind 75% thereof if its initial concentration is 5.5 mg/l.

The algae with mucilage bind all the silver if its initial concentration is 1.1 mg/l and bind 20% thereof if its initial concentration is 55 mg/l.

The rapid accumulation of silver by the algae can be explained by two phenomena. Firstly, by chelation outside the microalgae by the abundant mucilage, consisting of polymers of sugars and derivatives, which has the property of chelating cations. Secondly, by active and/or passive incorporation of the silver inside the algae via channels or transporters. The numerous transmembrane ion channels for example perform nonselective passive transport of Na⁺ ions, K⁺ ions or possible monovalent metals such as Ag⁺, to the cytoplasm, all the more readily if the concentration gradient is high.

Thus, the concentrating of silver by Coccomyxa corresponds to adsorption on the mucilage and the parietal compounds, added to which is intracellular absorption via transmembrane ion channels and active transporters.

Cobalt

Experiments were carried out with algae having little or no mucilage, according to the same protocol as that described above for silver. The algae were exposed to initial cobalt concentrations of 19 μg/L, 0.94 mg/L and 4.8 mg/L.

The results are represented in FIG. 8(A-C).

Initial rapid adsorption, possibly coupled with absorption, is observed. It is, however, less marked for cobalt than for silver. The absorption then continues gradually over several days. The concentration of intracellular cobalt does not reach a maximum, but continues to increase more than a week after the beginning of the exposure.

The cobalt absorption mechanism therefore appears to be different than that of silver. The divalent Co²⁺ ions cannot in fact use the same ion channels as the Ag⁺ ions because of their larger ionic radius. It is likely that cotransporters, responsible for the absorption of divalent metals essential for cell growth (Fe²⁺, Mg²⁺, etc.) or active transporters, such as ATP-dependent ABC pumps known as transmembrane transporters of cadmium Cd²⁺, or Zn²⁺ ATPases, carry out the absorption of cobalt. This active transport would explain a moderate but long-lasting absorption.

Maximum Metal Concentrations Tolerated

The limiting doses supported by Coccomyxa actinabiotis were investigated by exposure of the algae to silver or to cobalt for one week (20 mg of microalgae (fresh mass) in 100 ml of deionized water containing Ag or Co ions at various concentrations).

The algae without mucilage are less resistant to silver metal toxicity than the algae with mucilage. The mucilage could prevent a rapid and strong afflux of metal into the cytoplasm at the beginning of exposure. By forming an ion-binding barrier, it limits diffusion to the transmembrane channels and rapid and massive importing of toxic metals. These metals would then be moderately and gradually absorbed and could be stored in forms not aggressive for the cell. Coccomyxae actinabiotis without mucilage do not survive at silver concentrations greater than 250 μg/L, whereas Coccomyxae actinabiotis with mucilage withstand extracellular silver concentrations of at least 50 mg/L.

The cobalt intolerance threshold is greater than Co²⁺ concentrations of 50 mg/l and 800 mg/l for Coccomyxa actinabiotis without mucilage and with mucilage, respectively. No notable effect was observed on the microalgae cultured for one week under these conditions.

Maximum Metal Concentrations Bound

The algae can bind much higher amounts of Ag and of Co than what might be expected by passive binding, in particular owing to their synthesis of abundant mucilage and to active mechanisms of incorporation and detoxification.

Generally, metals are toxic for organisms.

Silver is generally toxic for organisms at 10⁻⁷-10⁻⁵ mol/l (Ratte H T, Environ. Toxicol. Chem. 18: 89-108, 1999).

Radioresistant organisms such as Deinococcus radiodurans do not grow in the presence of cobalt at 10⁻⁴ mol/l (John et al., 2000, above). However, the Coccomyxa actinabiotis microalga does grow in the presence of 10⁻⁴ mol/l of cobalt.

Coccomyxa actinabiotis algae brought into contact, in a BBM culture medium diluted 10 times, with an equivalent of 6.7×10⁻⁴ mol of silver per gram of alga bind up to 43.7 mg of silver per gram of fresh mass, i.e. approximately 450 mg/g of dry mass.

This amount of silver bound is much higher than those reported in the literature. The literature reports silver concentrations in terrestrial plants of between 0.01 and 150 μg of silver/g of dry mass, i.e. approximately 0.001 to 15 μg of silver/g of fresh mass, and silver concentrations in algae of between 3 μg/g and 7 mg/g of dry mass, i.e. approximately 0.3 μg to 0.7 mg of silver/g of fresh mass. The amount of silver bound by Coccomyxa actinabiotis is even greater than the amount of silver that can be accumulated by bacteria which exhibit extremely high tolerance to silver: silver-hyperaccumulating bacteria accumulate up to 300 mg of silver/g of dry mass (Ratte, 1999, above; Charley et al., Arch. Microbiol. 123: 239-244, 1979). Furthermore, the most highly silver-accumulating organisms are not radioresistant.

Coccomyxa actinabiotis algae brought into contact for 45 days with an equivalent of 3.8×10⁻⁴ mol of cobalt per gram of alga in a BBM nutritive medium diluted 10 times bind 12.1 mg of metal per gram of fresh biomass. In 6 days, algae brought into contact with an equivalent of 1.0×10⁻³ mol of cobalt per gram of alga in a BBM nutritive medium diluted 10 times bind 1.53 mg of metal per gram of fresh biomass. These values are much higher than those reported in the literature. The average concentration of cobalt in plants varies between 0.1 and 115 μg/g of dry mass, i.e. approximately 0.01 to 11 μg/g of fresh mass, and cobalt-hyperaccumulating plants bind up to 4.3 mg thereof/g of dry mass (Bresson et al., Toxicologie nucléaire, environnementale et humaine [Nuclear, environmental and human toxicology], publisher Lavoisier 2009, editions TEC and DOC. Chap. 29 Cobalt, p. 553-573), i.e. approximately 0.4 mg/g of fresh mass. The most highly cobalt-accumulating organisms cited in the literature are not radioresistant.

EXAMPLE 4 Fixing of Carbon 14

Carbon 14 constitutes, with tritium, the principal radioactive pollutant discharged into the environment by nuclear plants (8 to 25 GBq of ¹⁴C/year in the liquid waste from EDF plants). ¹⁴C waste has been regulated only since 2008. For the moment, there is no specific ¹⁴C treatment. The regulations in terms of ¹⁴C in liquid radioactive waste vary from one plant to another. The threshold is set at 150-400 GBq for an EDF power station (ordered 2008). ¹⁴C is discharged into liquid effluents essentially in the form of ¹⁴CO₂, carbonate (¹⁴CO₃ ²⁻ or H¹⁴CO₃ ⁻) ¹⁴CO and a small percentage in unidentified organic form. Like all plants, the Coccomyxa green microalga is capable of incorporating carbon in the form of ¹²CO₂ via photosynthesis. The isotopic discrimination of ¹⁴C with respect to ¹²C is negligible in biological processes. However, contrary to what might be deduced therefrom, the fixing of ¹⁴C by the Coccomyxa actinabiotis microalga is not at all obvious. For example, the alga does not fix ¹⁴C at pH 8, but fixes it at pH 7.

Furthermore, the fixing of ¹⁴C via biological processes such as photosynthesis can be carried out only with live photosynthetic biomass.

The impact of ¹⁴C on the physiology of the microalgae and the parameters which make it possible to carry out and optimize its accumulation by these microorganisms were determined.

¹⁴C is assayed in the algae and in the water by liquid scintillation. The microalgae are brought into contact with ¹⁴C in the form of carbonate CO₃ ²⁻ in acid-base equilibrium with HCO₃ ⁻ and CO₂ (pKa of 6.4 and 10.3) and also in the form of acetate.

Impact of ¹⁴C on the Physiology of the Microalgae

The impact of the presence of ¹⁴C on the physiology of the algae was evaluated by monitoring the growth of the cells. The presence of ¹⁴C alone, up to 20 000 Bq/l, has no impact on growth, compared with that of a culture not exposed to ¹⁴C.

Cells placed in culture in water from a spent nuclear fuel storage pool, containing gamma emitters and enriched with 300 000 Bq/l of ¹⁴C, also experienced growth of their population for 8 days.

Parameters Influencing the Incorporation of ¹⁴C by the Algae

Chemical Form of ¹⁴C

The algae incorporate the carbon dioxide inorganic form in equilibrium with the hydrogen carbonate, just as they do the acetate organic form. The CO₂ is incorporated into the cells via photosynthesis (6 CO₂+12 H₂O+hν→C₆H₁₂O₆+6 O₂+H₂O). The acetate is used by the metabolism in the form of acetyl coenzyme A and in particular in the main energy cycle of the cell (Krebs cycle).

pH

The influence of the pH on the incorporation of ¹⁴C in hydrogen carbonate form was studied in the range 6.9 to 8.5. The optimum pH is 6.9 and allows the incorporation of from 80 to 90% of the ¹⁴C present in the medium when the containers in which the bringing into contact is carried out are hermetically closed. At this pH, 25% of the ¹⁴C is in the form of dissolved CO₂. It is necessary to close the containers in order to avoid rapid degassing of the ¹⁴CO₂ in equilibrium with H¹⁴CO₃ ⁻.

The amount of ¹⁴C incorporated by the algae is low at pH 7.5 (a few %) and zero at pH 8.5.

Initial Conditions for Culturing the Microalgae

The physiological state of the cells at the moment they are brought into contact with the ¹⁴C has an influence on the rate of incorporation of the ¹⁴C. The microalgae incorporate the ¹⁴C much more rapidly when they have been cultured beforehand in a medium richer in nutrients (BBM medium diluted two-fold preferable to a BBM medium diluted ten-fold). Thus, the optimum time for bringing the algae into contact with a medium containing 20 000 Bq/l of ¹⁴C is 50 h if the culture is derived from a BBM medium diluted ten-fold, whereas it is reduced to 3 h if the culture is derived from a BBM medium diluted two-fold.

Density of the Algae

The amount of ¹⁴C incorporated in a given period of time increases with the density of the algae in the range 5×10⁶ to 15×10⁶ cells/ml. By way of example, 50 h are necessary for an algal population with an initial cell density of 5×10⁶ cells/ml to incorporate 70% of the amount of ¹⁴C present in the medium (20 000 Bq/l), whereas 7.5 h are sufficient for an algal population with an initial cell density of 15×10⁶ cells/ml to incorporate 90% thereof.

Illumination Intensity

The ¹⁴C incorporation experiments were carried out under weak illumination (50 to 70 μmol of photons/m²/s). However, the photosynthetic activity increases with the illumination, to reach a maximum starting from 500 μmol of photons/m²/s. The measurement of the maximum photosynthetic activity of the algae, carried out with an illumination of more than 500 μmol of photons/m²/s, shows that the algae are capable of consuming much more ¹²CO₂ under stronger illumination (at least 30 times more than at the light intensities at which the ¹⁴C incorporation experiments were carried out).

¹⁴C Concentration

Regardless of the initial concentration of ¹⁴C in the range 2000 to 20 000 Bq/l (which corresponds to the concentrations present in the water from the pools of certain nuclear plants), at pH 6.9, the algae rapidly and strongly incorporate the ¹⁴C. The maximum rate of ¹⁴C incorporation by a population of algae derived from a culture in BBM medium diluted two-fold is 20 Bq/h/10⁶ cells for a ¹⁴C concentration of 6000 Bq/l and is 60 Bq/h/10⁶ cells for a ¹⁴C concentration of 20 000 Bq/l. The amounts incorporated are about 80-90%.

The incorporation of ¹⁴C in hydrogen carbonate form, at pH 6.9, by a culture of Coccomyxae actinabiotis derived from a BBM medium diluted two-fold, at the initial concentration of 5×10⁶ cells/ml, illuminated with 70 μmol of photons/m²/s, was measured. The ¹⁴C concentration is 6000 Bq/l. After centrifugation of samples at the various times, the ¹⁴C content is analyzed in the pellet (algae) and in the supernatant. The results are illustrated by FIG. 9.

Optimum Operating Conditions for ¹⁴C Purification

pH 6.9

cells initially cultured in a medium rich in nutrients (BBM diluted two-fold)

cell density of 15×10⁶ cells/ml

intense illumination (500 μmol of photons/m²/s)

contact time: a few hours

Performance Levels

90% decontamination

Coccomyxae actinabiotis can therefore be used to decontaminate the carbon 14 contained in water resulting from nuclear plants: 90% decontamination in 3 to 7 hours, under optimized conditions.

EXAMPLE 5 Binding of Uranium

The algae according to the invention can also be used to take up uranium and transuranium elements.

Coccomyxae actinabiotis microalgae harvested from a round-bottomed culture flask and washed three times with Milli-Q water in order to remove the culture medium were brought into contact, with shaking and at a concentration equivalent to 60 mg of algae by fresh mass/100 ml of solution, with a solution of uranyl nitrate having a concentration of 10⁻⁸ mol/l. In 26 h, the algae bind 45% of the amount of uranium initially present in solution. Under these conditions, the factor of uranium bioconcentration by the Coccomyxae actinabiotis algae is 1300 l/kg of fresh mass, i.e. approximately 13 000 l/kg of dry mass. The bioconcentration factors reported in the literature are about 120-140 l/kg for freshwater or seawater algae (Paquet et al., Toxicologie nucléaire, environnementale et humaine [Nuclear, environmental and human toxicology], publisher Lavoisier 2009, editions TEC and DOC. Chap. 23 Uranium, p. 411-443).

EXAMPLE 6 Decontamination of Storage Pool Water by Means of Coccomyxae actinabiotis Via In Situ Action

The Coccomyxae actinabiotis microalgae were placed on horizontal supports in a fuel element cooling pool of a nuclear plant. This pool is filled with water having an average pH of 5.5, an average conductivity of 1.2 μS/cm, and an average temperature of 25° C., and contains radioactive metal elements owing to the parts which are stored therein. The pool is in contact with the ambient air and is illuminated by neon light. The light intensity at the surface of the water is 200 lux. Depending on the parts stored in the pool, the dose rate at the level of the algae ranges from about 100 μGy/h to a few tens of Gy/h. Under these conditions, Coccomyxae actinabiotis is capable of colonizing the supports on which it is placed, and can live thereon and reproduce thereon for years. The dose rate can sporadically reach a few hundred Gy/h.

The total activity and also the nature and the activity of each γ-emitter present were determined by performing counting by γ-spectometry on samples of 50 ml of water and of 100 mg of algae sampled from the supports. The results obtained after a residence time of 5 years are given in table IV below.

TABLE IV Activity (Bq) of 1 ml Activity (Bq) of 1 g Radionuclide of water of microalgae ⁵¹Cr 1.73 10543 ⁵⁴Mn 0.15 168 ⁶⁰Co 0.64 24733 ⁶⁵Zn 0.13 363 ^(108m)Ag <limit of detection = 0.02 556 ^(110m)Ag 0.006 2671 ¹²⁴Sb 0.87 149

These results show that the algae are approximately 10 000 times more active than the water in which they live: the radioelements are actually concentrated on the outside and on the inside of the microalgae.

The silver in particular has virtually disappeared from the water. The cobalt and the chromium were very strongly concentrated.

EXAMPLE 7 Decontamination of Storage Pool Water by Means of Coccomyxae actinabiotis Via In Situ Action and Comparison with Conventional Methods

An experiment in which water from a radioactive element storage pool, with a total volume of 361 m³, was decontaminated was carried out in situ using the Coccomyxa actinabiotis microalga in suspension in the pool to be depolluted. At t=14 days, two safety rods from a reactor were introduced into the pool. These rods release silver 110m. The water is usually purified by ion exchange resins which bind the radionuclides. At t=30 d, the purification by the resins was stopped. From t=35 d to t=56 d, the water was purified by uptake of the radionuclides by the algae in suspension in the pool. These algae, and also the radiological activity collected, bind to micropore filters (50 mm diameter, 60 mm height) installed in a mobile pool robot at the surface. The filters, which are highly active, are changed every 2 days.

FIG. 10 shows the concentration of silver 110m in this pool as a function of time, expressed in number of days. The decrease in the ^(110m)Ag concentration observed between 35 and 56 d is due to its uptake by the algae present in the pool and which bind to the filters.

Starting from t=60 d, the ion exchange resins were put back into operation. FIG. 10 shows purification of the radioelements with similar efficiency whether it is carried out with the microalgae or with the ion exchange resins. In total, during this experiment, 740 MBq of γ emitters (of which 470 for ^(110m)Ag, 180 for ¹²⁴Sb and 90 for ⁶⁰Co) were removed from the pool by the algae in 21 days.

EXAMPLE 8 Decontamination of Nuclear Plant Water by Means of Coccomyxae actinabiotis Via a Transported Method

Water originating from a spent nuclear element storage pool was brought into contact with Coccomyxae actinabiotis(50 ml of water in contact with 9×10⁸ cells, i.e. 140 mg of algae by fresh mass, originating from culture and rinsed beforehand three times with Milli-Q water). The water initially contains the radionuclides listed in table V below.

The assaying of the water and of the algae 24 h after the bringing into contact shows an elimination of 92% of the γ-activity of the water in 24 h.

Table V below indicates the initial composition of the water, according to the total γ-activity measured and the γ-activity measured per radionuclide, and the percentage of γ-emitting radionuclides bound by the algae at 24 h.

TABLE V Total ⁵⁴Mn ⁶⁰Co ^(110m)Ag ¹³⁷Cs ²³⁸U Initial γ-activity in the 161.9 9.2 42.4 22.7 66.5 21 water (Bq/l) % binding 92 100 72 100 100 95

The binding is therefore 100% for silver 110m, manganese 54 and cesium 137, 72% for cobalt 60, and 95% for uranium 238.

EXAMPLE 9 Control of the Proliferation of the Microalgae

The microalgae are photosynthetic. They need light to perform photosynthesis and produce their organic matter. In the highly nutrient-depleted medium which corresponds to the various media and effluents from nuclear power stations, their growth can therefore be controlled by illumination. In order for them to grow at a given place, it is sufficient to illuminate them. In the pool where they were uncovered, they proliferate preferably close to the light sources. When they are placed in the dark for a period of 2 days to 1 month, their growth stops. Their growth can also be controlled by illuminating them with a light which allows little or no photosynthesis, for example with a yellow-green-colored non-actinic lamp.

Filtration of the water makes it possible to take up the algae in suspension in the water, and to control the growth thereof. 

The invention claimed is:
 1. An isolated single-cell green alga of the Coccomyxa genus, characterized in that it belongs to the Coccomyxa actinabiotis species defined by the presence, in the 18S ribosomal RNA-ITS1-5.8S rRNA ITS2-26S rRNA genes, of a sequence exhibiting at least 95% identity with the sequence SEQ ID NO:
 1. 2. The isolated single-cell green alga as claimed in claim 1, which is the Coccomyxa actinabiotis strain deposited on Jun. 25, 2009 with the Culture Collection of Algae and Protozoa (CCAP) under number CCAP 216/25.
 3. A method for taking up at least one element chosen from the metals Ag, Co, Cr, Zn, Mn, Sb, Ni, Fe, Cs, actinides and lanthanides, and the radioisotopes ¹⁴C and ³H, from an aqueous medium containing said metal or said radioisotope in solution, characterized in that said uptake is carried out by incubating the isolated single-cell green algae of the Coccomyxa genus of claim 1 in said aqueous medium.
 4. A method for taking up at least one element chosen from the metals Ag, Co, Cr, Zn, Mn, Sb, Ni, Fe, Cs, actinides and lanthanides, and the radioisotopes ¹⁴C and ³H, from an aqueous medium containing said metal or said radioisotope in solution, characterized in that said uptake is carried out by incubating the isolated single-cell green algae of the Coccomyxa genus of claim 2 in said aqueous medium.
 5. The method as claimed in claim 3, characterized in that said aqueous medium is a radioactive medium.
 6. The method as claimed in claim 5, for taking up a metal chosen from Ag, Co, Cr, Zn, Mn, Sb, Ni, Fe, Cs, actinides and lanthanides, characterized in that said metal is in the form of a radioactive isotope, or in the form of a mixture of isotopes.
 7. The method as claimed in claim 3, characterized in that the isolated single-cell green algae of the Coccomyxa genus is a Coccomyxa actinabiotis species and the growth of the isolated single-cell green algae of the Coccomyxa actinabiotis species is controlled by controlling the illumination of said aqueous medium.
 8. The method as claimed in claim 7, characterized in that the element taken up is a metal, and in that it comprises a step of recovering said metal from the algae.
 9. A method for decontaminating a radioactive aqueous medium containing at least one element chosen from the metals Ag, Co, Cr, Zn, Mn, Sb, Ni, Fe, Cs, actinides and lanthanides, and the radioisotopes ¹⁴C and ³H, comprising incubating the isolated single-cell green algae of claim 1 in the radioactive aqueous medium.
 10. A method for decontaminating a radioactive aqueous medium containing at least one element chosen from the metals Ag, Co, Cr, Zn, Mn, Sb, Ni, Fe, Cs, actinides and lanthanides, and the radioisotopes ¹⁴C and ³H, comprising incubating the isolated single-cell green algae of claim 2 in the radioactive aqueous medium. 